Introduction: Until recently, interpretation of neural effects on the heart was confined to the effects of centrally derived extrinsic sympathetic and parasympathetic inputs. However, it is now known that neurocardiac control is more complex due to an extensive independent network of neurones constituting a ‘little brain’, that are capable of modulating different cardiac functions. 1 These intrinsic neurons occur in discrete regions on the surface of the heart and within epicardial fat pads where they form “ganglionic plexuses” and function as integrative circuits acting throughout the heart. 2 A recent study in the rabbit shows that the locations of these ganglionic plexuses are similar to other species and are primarily located on the heart hilum, around the pulmonary arteries and right caudal vena cava and on the conus arteriosus. 3 In the rabbit, information pertaining to the functional effects of these ganglionic plexuses and their neurotransmitter content is absent. The aims of this study were to 1) assess the neurotransmitter phenotype, and 2) investigate the functional role of the ganglionic plexuses in the rabbit heart. Methods: New Zealand White rabbit hearts (1-3kg) were used. After sedation (ketamine, 10mg/kg [Ketaset]; medetomidine hydrochloride [Sedator], 0.2mg/kg; butorphanol, 0.05mg/kg [Torbugesic]; im), animals were heparinised (1000 IU) and killed with sodium pentobarbitone (160mg/kg, iv). Aim 1: The left and right side of the heart was cannulated and the heart flushed from blood using 0.1M phosphate buffered saline (4oC) and inflated with 20% gelatine. Following removal of material overlaying the heart hilum, hearts were fixed with 4% paraformaldehyde, and treated with hyaluronidase (0.5%) then briefly stained for aceylcholinesterase (AChE) to confirm ganglia location. The whole mounts were then micro-dissected from the heart and treated with appropriate blocking buffers and incubated with primary antibodies raised against choline acteyltransferase (ChAT), tyrosine hydroxylase (TH), neuronal nitric oxide synthase (nNOS) and protein gene product 9.5 (PGP) for 48 hours (4oC). Visualisation of each neurotransmitter target was achieved following incubation with appropriate secondary antibodies tagged to specific Alexa Fluor fluorescent dyes. Aim 2: Hearts were perfused in constant flow Langendorff mode and instrumented to record left ventricular pressure, perfusion pressure, atrial and ventricular electrograms. Nicotine was applied to different regions of the heart that contained ganglionic plexuses (0.2 to 4 mg in 10 to 100 μL saline) and heart rate and atrio-ventricular conduction measured. Tests were carried out in sinus rhythm and during atrial pacing in the absence and presence of muscarinic, nicotinic and beta-adrenoreceptor blockade.Results: Aim 1) Cell bodies and epicardial nerves were positively labelled for AChE throughout the heart hilum, around the pulmonary arteries and right caudal vena cava and on the conus arteriosus. ChAT was the primary neurotransmitter found in the intrinsic cardiac nerve plexus with lesser amounts of cell bodies and nerves immuno-reactive for TH and nNOS. Co-location with PGP confirms the structures positively stained were neuronal in nature. Aim 2) Nicotine applied locally to regions containing ganglia elicited either a bradycardia, tachycardia or brady/tachycardia that was either dependent on cholinergic or adrenergic nerves. In addition to these heart rate effects, nicotine primarily produced a delay in atrio-ventricular conduction. Ganglionic plexus with specific cardiac actions were found. Conclusion: These are the first data that describe the neurotransmitter content and functional effects of activation of different ganglionic plexuses in the rabbit heart.